Process for producing synthetic tryptophane



United States Pater Of'ice 3,019,232 PROCESS FOR PRODUCING SYNTHETICTRYPTOPHANE Setsuji Sakurai, Fujisawa City, Yoshioki Komachiya, Tokyo,and Tamio Ito, Kawasaki City, Japan, assignors to Ajinomoto Co., Inc.,Tokyo, Japan No Drawing. Filed Nov. 24, 1959, Ser. No. 855,015 Claimspriority, application Japan Dec. 13, 1958 7 Claims. (Cl. 260-319) Thepresent invention relates to a process for producing tryptophan, andmore particularly to a process of synthesizing tryptophan, from easilyavailable compounds.

Tryptophan is one of the essential amino acids and has importantapplications in the fields of medicine and biochemistry.

It is practically impossible to obtain tryptophan by acid hydrolysis ofprotein, since tryptophan reacts with the glucose produced bydecomposition of carbohydrates contained in the raw material, to formdark coloured insoluble hurnin. The use of alkalies instead of acids forhydrolysis of protein is not convenient in an industrial operation.Enzymatic hydrolysis not only requires a very long time, but it alsodoes not proceed to completion and gives a low yield.

It is the main object of this invention to produce tryptophan from acomparatively cheap and easily obtainable compound and in a high yield.

We have found that the phenylhydrazone of u-ketoglutaric acid (I), whichhas recently become available at low cost, when subjected to indolcondensation in alcohol, forms ethyl [Z-carboethoxy-indolyl-(3)]-acetate(II). When the latter is further subjected to condensation with adiethyl oxalate, ethyl fl-[Z-carboxy-indolyl- (3)]-ethoxalyl-acetate(Ill) is obtained in a good yield. When the thus obtained compound (III)is heated in acetic acid in the presence of a catalyst,p-toluenesulfonic acid, there is formed2,3-indo-3,4-pyrone-(2)-carboxylic acid-(6) (IV) but notfi-[2-carboxy-indolyl-(3)]-pyruvic acid (V).

We have further found that said B-[Z-carboxy-indolyl- (3)]-pyruvic acid(V) can be obtained in a high yield, if said2',3-indo-3,4-pyrone-(2)-carboxylic acid-(6) (IV) is treated with anaqueous solution of a caustic alkali.

l H H 3,019,232 Patented Jan. 30, 1962 T-CHrC OC O OH NlCO 011 In orderto carry out the last mentioned reaction,2,3'-indo-3,4-pyrone-(2)-carboxylic acid-(6) is suspended in an aqueoussolution of about 20% caustic alkali and the suspension is agitated. Thereaction is completed in 6090 minutes at room temperature and in 5-6minutes at C. The compound formed thereby may be separated from thereaction mixture by cooling and acidifying the latter, extracting thecompound with a solvent such as ethyl ether, ethyl acetate and the likefrom said reaction mixture and evaporating the solvent from the extract.Almost pure fi-[Z-carboxy-indolyl- (3) ]-pyruvic acid is obtained.

EXAMPLE 1 19.5 g. of 2,3-indo-3,4-pyrone-(2)-carboxylic acid (6) areadded to an aqueous solution of 29 g. of caustic potash in cc. of Waterand the mixture is agitated at 25 C. The compound dissoives completely.After agitation has been continued for 1 hour, 20% sulfuric acid isadded drop by drop to the solution to adjust its pH to 1.5 while it isbeing cooled with ice. The acidified solution is extracted With ethylether 5 times. Ether is evaporated from the combined extracts at reducedpressure. The residue Weighs 15 g. After recrystallization fromacetone-benzene, 11 g. of crystallized fi-[Z-carboxyindolyl(3)]-pyruvicacid are obtained.

The melting point of the compound (V) cannot be determined, because thecompound is easily converted to 2,3'indo-3,4-pyrone-(2)-carboXylic acid(6) at elevated, temperature. The results of an analysis compare withvalues calculated for C H O N as follows:

Calculated: C, 58.30; H, 3.67; N, 5.67. Found: C,

Quantitative determination of fl-[Z-carboxy-indolyh (3)1-pyruvic acid bymeans of 2,4-dinitrophenyl-hydrazone shows a yield of 82%. For thispurpose 12 cc. of the reaction mixture acidified to pH 1.5 With sulfuricacid are Withdrawn as a sample and 200 cc. of a 0.2% aqueous solution of2,4-dinitrophenyl-hydrazine hydrochloride are added 1.2 g. of hydrazonecrystals are formed. When recrystallized from water-ethanol, 0.6 g. ofcrystals having a melting point of 227228 C. are obtained. The resultsof an analysis compare with values calculated for C H O N as follows:

Calculated: C, 50.59; H, 3.07; N, 16.39. Found: C, 50.17; H, 3.54; N,15.83.

We have further found that B-[Z-Earboxy-indolyl-(Bflpyruvic acid (V)when reacted with ammonia and then hydrogenated is transformed intoB-[Z-carboxy-indolyl- (3)]-u-aminopropionic acid (VI).

Table 1 Initial Temperpressure, ature, Yield,

atrn. C percent EXAMPLE 2 19.5 g. of 2,3'-indo-3,4-pyrone-(2)-carboxylicacid (6) (IV) are added to a solution of 29 g. of caustic potash in 100cc. of water and the resulting mixture is agitated for 1 hour at 25 C.To the reaction mixture containing fi-[2-carboxy-indolyl-(3)]-pyruvicacid (V) are added 200 cc. of 28% ammonia water, 100 cc. of water and g.of Raney nickel, as catalyst, and it is subjected to hydrogenation underan initial pressure of 50 atm. at 50 C. The reaction is completed in 90min. Nickel is filtered off and the ammonia is expelled from thereaction mixture. The solution is concentrated to about 100 cc., and itspH is adjusted to 2.5 with 20% sulfuric acid. The precipitated crystalsare washed with hot water to remove potassium sulfate and further washedwith 100 cc. of ethanol, giving 21 g. of crystals ofB[2-carboxy-indolyl- (3)1-Ot-BIHIHOP1'OP1OHIC acid (VI). Itsdecomposition point is 220222 C. and the yield is 95%. Afterrecrystallization from water, the decomposition point is 226228 C. onemole of water of crystallization is present. The analysis resultcompares with the calculated values for C H O N .H O as follows:

Calculated: C, 54.20; H, 5.27; N, 10.53. Found: C, 54.04, H, 5.28, N,10.90.

The benzoyl derivative which is obtained from compound (VI) in 70% yieldhas its decomposition point at 236-237 C. and its analysis compares withthe calculated values for C H O N as follows:

Calculated: C, 64.77; H, 4.58; N, 7.95. Found: C, 64.70; H, 4.45; N,7.99.

The compound (VI) may be converted tofi-[Z-carboxyindolyl-(3)]-u-uramino-propionic acid (VII) with highyield. 32 g of B-[Z-carboxy-indolyl-(B)]-a-amino-propionic acidmonohydrate and 43.2 g. of urea are heated in 360 cc. of water in thepresence of 86 g. of barium hydroxide to the boiling temperature for 5hours. The reaction mixture is filtered while hot. The filtrate, afterbeing cooled, is adjusted to pH 2 with hydrochloric acid. Crystals whichprecipitate from the solution while standing over night, are washed withwater and dried. 34.2 g.

of crude fl-[2-carboxy-indolyl-(3)l-u-uramino-propionic acid (VII) arethus obtained. Instead of barium hydroX ide, sodium hydroxide may beused as a condensing agent. However, the former gives a compound ofhigher purity than the latter. The following table illustrates therelationship between reaction conditions and yields. The figures inbrackets show molar proportions.

Table 2 Oom- Boilpound NaOH, Ba(OH)2, Ure H1O, ing Yield, (VI), g". g.g. g. cc. time, percent hrs.

32. 0 4. 8 0 36. 0 360 5 65. 9 (1.0) (1. 0) (5.0) 32.0 11. 1 0 36. 0 3605 85. 8 (1.0) (2.3) (5.0) 32. O 16. 8 O 36. 0 360 5 64. 5 (1. O) (3. 5)(5. 0) 32. 0 4. 8 3i. 0 36. 0 300 5 87. 9 (1.0) (1.0) (0.9) (5. 0) 32. 04. 8 34. 0 36. 0 600 5 84. 4 (1. 0) (1. 0) (0. 9) (5.0) 32. 0 9. 6 34. 036. 0 600 5 86. 6 (1. 0) (2.0) (0.9) (5.0) 32. 0 0 72. 0 36. 0 360 4 84.2 (1.0) (1.9) (5. 0) 32. 0 0 86. 0 43. 2 360 5 91. 9 (1. 0) (2. 28) (6.0) 32.0 0 72. 0 25. 2 1,260 5 48. 6 (1. 0) (1.9) (3. 5) 32. 0 0 72. 036. 0 1, 800 5 51. 3 (1.0) (1.9) (5.0) 32.0 0 72. 0 50. 3 2, 515 5 78. 5(1. O) (1. 9) (7. 0)

We have further found that decarboxylation of comp und (VII) takes placewhen the same is heated in hydrochloric acid containing an alcohol, and3-(hydanto- 5-yl-methyl)-indol (IX) is obtained in a good yield. Thecompound (IX) is converted almost quantitatively to tryptophan (X), whenthe former is hydrolyzed in an aqueous solution of ammonia or causticsoda.

3.08 g. of B-[2-carboxy-indoly1-(3)J-u-uraminorQ- pionic acidmonohydrate are added to a solution consisting of 15 cc. of ethanol and30 cc. of concentrated hydrochloric acid and the mixture is held at86-90" C. for 30 minutes. After cooling, crystals are precipitated whichweigh 0.16 g. and have a decomposition point of 282- 284 C. (thehereinafter discussed Compound VIII). A portion of the filtrate obtainedafter removal of the crystals is evaporated at reduced pressure todryness. The

residue is recrystallized from 10 cc. of 50% aqueous ethanol and 0.97 g.of 3-(hydanto-5'-yl-methyl)-indol (IX) having a melting point of 2l7.5C. are obtained.

The Compound VIII is decarboxylated by heating in parafiin to 290 C. Theresidue after removal of the parafiin is hydrolyzed with an aqueoussolution of caustic The remainder of the aforementioned filtrate, fromsoda, and tryptophan (X) is obtained in a yield of apwhich 0.16 g. ofcrystals (VIII) have been separated, is proximately 50%. However, if 1mole of Compound mixed with 100 cc. of 28% ammonia water and kept atVIII is boiled in an aqueous solution of 2 moles of caustic 170 C. for 2hours. The ammonia water is then soda for 6 hours, it is transformedinto the Compound evaporated at reduced pressure. 1.93 g. of crude tryp-VII and if it is treated with an aqueous solution of 6 moles tophanhaving a decomposition point of 262-263 C. are of caustic soda, theCompound VI is formed almost obtained. Its purity is determined as 90%by bioassay. quantitatively.

Accordingly, the yield of the Compound IX produced The Compound IX canbe easily converted to tryptoby decarboxylation from the Compound VII ismore phan (X) in the usual manner. For example, 22.7 g. than 1.94 g.which corresponds to more than 85% yield. of3-(hydanto-5'-yl-methyl)-indol (IX) are added to a Although thedecarboxylation may be performed in solution of 14.5 g. of causic sodain 280 cc. of water hydrochloric acid only, the yield of Compound IX isthen and the mixture is heated to 150 C. i-3 C. for 30 minapproximatelyhalf of that of Compound VI-II. On the utes. After cooling, the reactionmixture is evaporated contrary, when decarboxylation is carried out inhydroto about 100 cc., decolorized with activated carbon, mixed chloricacid containing an alcohol, the yield of Comwith 50 cc. of ethanol,adjusted to a pH below 6.0 with pound IX increases substantially, When amixture of acetic acid and left standing Overnight in a cOld room. 1volume of ethanol and 2 volumes of hydrochloric acid The precipitatedcrystals are separated from the mother is used, the yield reaches 85%.However, the use of too liquor, washed with water and ethanol, anddried. The much alcohol results in the predominant formation of CrystalsObtained Weigh and are 50 P that Compound VIII. Methanol, ethanol,isopropanol or like recrystallization is almost unnecessary. They have adealcohols may be used. Instead of hydrochloric acid, sulcompositionpoint of 283-4 C. Since 2.5 g. of tryptofuric acid may be used.Phosphoric acid is less elfective 2 1911311 can be recovered from themOthBf liquor y resin and such weak acids as acetic acid areineffective. The treatment, yiel f t e hydrolyzing proceduredecarboxylation depends on the kind of solvents used, but amounts to92%. essentially proceeds at a temperature above 80 C. Too What We claimis: high a temperature, however, causes the decomposition of 1. In aprocess of producing tryptophan, the steps of Compound IX. Therespective yields of Compounds IX treating 2,3- indo-3,4-pyrone-(2)-carboxylic acid-(6) and VIII with various combinations ofacids and alcohols With an aqueous solution of caustic alkali, whereby3-[2- at diflerent temperatures are seen in Table 3. In all Y' Y-U)l-pyruvic acid is formed; reacting reactions, 3.08 g. offl-[Z-carboxy-indolyl-(B)]-aaid B-[2-c rboxy-in 1y1-(3)l-pymvic acid ithamm uramino-propionic acid monohydrate (VII) were used a nia; andhydrogenating the reaction product obtained in a starting material. Theyields of Compound IX are calthe presence of a nickel hydrogenationcatalyst to form culated from that of tryptophan (X), assuming thelatter fl-[2-carboxy-indolyl-(3)-]-a-amino-propionic acid. to beobtained from Compound IX without loss. 2. In a process of producingtryptophan, the steps of Table 3 Yield of (IX) Yield of (VIII) Alcohol(or Temperwater) Cc. Acid 00. ature c o.) (0.) (Per- 0. (Percent) cent)15 Gone. 1101.. 30 105405 0.69 30 1.60 59 15 do.-- 30 86- 90 0. 92 1.2386- 90 1.40 01 0. 79 29 84- 85 1.83 80 0.52 19 86- 90 1. 94 85 0.10 686- 90 0. 53 23 1.30 51 90 95 0. 34 15 1.00 86- 90 0.83 30 1.30 50 Dos5- 90 0. s0 35 1.42 52 Metl1an0l 15 85- 90 1.85 81 0. 34 14 D0 s2- 850.85 37 1.34 49 Isopropanol 15 86- 90 0.62 27 1.31 48 D 22.5 do 22.5 80-90 0.50 22 1.45 53 15 33% H2SO4 30 se- 90 1.83 80 0.27 10 15 33% H roi.30 80- 90 0.46 20 0.13 5

The compound VII is heated in solution with an acid heating[2-carboxy-indolyl-(3)J-a-amino-pmpionic acid such as hydrochloric acidor sulfuric acid with or withand urea in the presence of a condensingagent selected out an alcohol, Compound VIII is obtained as a byfrom thegroup consisting of alkali metal and alkaline product together withCompound IX, the former being inearth metal hydroxides to formfl-[Z-carboxy-indolylsolub e While the latter is soluble in the reactionmixture. (3)]-a-uramino-propionic acid; heating the latter acid to Thefact that the dissolved compound is correctly reprean elevatedtemperature between about 80 C. and 106 sented by Formula IX is provedby elementary analysis C. in aqueous solution in the presence of astrong minand infrared spectrum which agree with those of Comeral acid,whereby 3-(hydanto-5'-yl-methyl)-indol is pound IX. Furthermore, thefact that the precipitated formed. crystals are the compound of FormulaVIII is proved by 3. A process as set forth in claim 2, wherein a lowerelementary analysis, decomposition point, and acid value alkanol isadded to the aqueous solution in which said which respectively agreewith those of Compound VIII. fi-[2-carboxy-indolyl -(3)]-a-uramino-propionic acid is Decomposition point: 282-4 C. heated in the presenceof the lower alkanol.

Elementary analysis (C H O N Calculated: C, 4. A process of producingtryptophan which com- 57.14; H, 4.06; N, 15.38. Found: C, 57.48; H,4.00; N, prises treating 2,3-indo-3,4-pyrone-(2)-carboxylic acid- 15.37.(6) with an aqueous solution of caustic alkali to form Acid value:Calculated: 205. Found 204. 8-[2-carboxy-indo1yl-(3)J-pyruvic acid;reacting said ,8-

7 [2-carboxy-indolyl-(3)J-pyiuvic acid with ammonia; subjecting thereaction product obtained to hydrogenation in the presence of a nickelhydrogenation catalyst at a temperature of at least 35 C. and hydrogenpressure of between about 15 atmospheres and 60 atmospheres, whereby 8-[2-carboxy-indolyl-(3 ]-a-amino-propionic acid is formed; heating thelatter acid and urea to an elevated temperature between about 80 C. and106 C. in the presence of a condensing agent selected from the groupconsisting of alkali metal and alkaline earth metal hydroxides to formfi-[2-carboXy-indolyl- (3)]-a-uramino-propionic acid; heating saidfi-[Z-carboxy-indolyl-(S)]-a-uramino-propionic acid in aqueous '8solution in the presence of a strong mineral acid, whereby3-(hydanto-5-yl-methyl)-indol is formed; and hydrolyzing the hydantoylgroup of said 3-(hydanto-5-yl-methy1)-indol to form tryptophan.

5. A process as set forth in claim 4, wherein said5-[2-carboxy-indoly1-(3)]-a-uramin0-propionic acid is heated in theadditional presence of a lower alkanol.

6. A process as set forth in claim 4, wherein said hydantoyl group ishydrolyzed in an alkaline medium.

7. A process as set forth in claim 4, wherein said hydrogen pressure isbetween substantially 30 and 50 atmospheres.

No references cited.

1. IN A PROCESS OF PRODUCING TRYPTOPHAN, THE STEPS OF TREATING2'',3''-3,4-PYRONE-(2)-CARBOXYLIC ACID-(6) WITH AN AQUEOUS SOLUTION OFCAUSTICALKALI, WHEREBY B-(2CARBOXY-INDOLY-(3))-PYRUVIC ACID IS FORMED;REACTING SAID B-(2-CARBOXY-INDOLYL-(3))-PYRUVIC ACID WITH AMMONIA; ANDHYDROGENATING THE REACTION PRODUCT OBTAINED IN THE PRESENCE OF A NICKELHYDROGENATION CATALYST TO FORMB-(2-CARBOXY-INDOLYL-(3)-)-A-AMINO-PROPIONIC ACID.